Thermoplastic shear localisation in titanium alloys during dynamic deformation

Cylindrical specimens (4 mm diameter and 4 mm height) of titanium alloy bar were given various heat treatments to provide a wide range of microstructures and mechanical parameters. These specimens were then subjected to high plastic strain at a large strain rate (103 s-1 ) during dynamic compression by a split Hopkinson bar at ambient temperature. The microstructures of the localised shear bands were examined by optical and transmission electron microscopy. The results show that there are two types of localised shear bands: deformed and white shear bands. A detailed observation reveals that there is no difference in the nature of the deformed and white shear bands, but they occur at different stages of localised deformation. It is found that there is a burst of strain, corresponding to a critical strain rate at which the white shear band occurs and no phase transformation occurs in the shear bands.

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2 alpha kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2 alpha kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2 alpha kinases might play similar roles and that PoPKR is the predominant kinase. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325-amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with. the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

Total and subcellular distribution of trace elements in the liver of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli)

Total and subcellular hepatic Zn, Cu, Se, Mn, V, Hg, Cd, and Ag were determined in a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). Except for higher fetal Cu concentration, all maternal elements were higher. Elements existed mostly in the cytosol of both animals except in the case of maternal Ag in the microsome and fetal Cu and Ag in the nuclei and mitochondria. In the maternal cytosol, Zn, Mn, Hg, and Ag were present in the high-molecular-weight substances (HMW); Se and V were present in the low-molecular-weight substances (LMW); Cu and Cd were mostly sequestered by metallothionein (MT). In the fetal cytosol, Zn, Se, Mn, Hg, Cd, and Ag were present in the HMW and V in the LMW, while Cu and Ag were mostly associated with MT. MT isoforms were characterized using the HPLC/ICP-MS. Two and four obvious peaks appeared in the maternal and fetal MT fractions, respectively. The highest elemental ion intensities were at a retention time of 7.8 min for the mother, and for the fetus the peak elemental ion intensities occurred at a retention time of 4.3 min, suggesting that different MT isoforms may be involved in elemental accumulation in maternal and fetal hepatocytosols. (C) 2003 Elsevier Ltd. All rights reserved.

Assembly of the subcellular parts of Bryopsis hypnoides Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20A mu mol/(m(2) s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation.

G蛋白在松类植物花粉管发育中的亚细胞定位及调控机制

G蛋白参与了哺乳动物内多种细胞信号途径，但其在植物花粉萌发和花粉管发育过程中的细胞学定位、生化特性及功能研究比较滞后，有关这方面的研究报道较少。在显花植物授粉受精过程中，具顶端极性生长特性的花粉管是雄性生殖单位的载体，也是研究细胞生长分子调控机理的理想体系。与被子植物相比，裸子植物具有生长周期长，花粉管生长缓慢、易分叉等特点，具有不同于被子植物花粉发育的独特发育模式。对于裸子植物花粉萌发和花粉管生长的调控机理，目前尚不十分清楚。本文以松类植物中比较有代表性的裸子植物青杆（Piceawillsonii）和白皮松(Pinus bungeana)花粉为试材，应用免疫分析和间接免疫荧光显微镜技术，结合药理学实验和FTIR手段，研究了异三聚体G蛋白和小G蛋白在花粉管细胞中的定位、生化特性及其在花粉管发育中的调控作用。结果如下： 应用Western Blotting技术和来自于抗哺乳动物中不同序列G蛋白O【亚基抗体，我们在白皮松花粉管中检测到一条分子量为40 kDa左右的蛋白。去污剂处理显示，该蛋白与质膜偶联。间接免疫荧光显微镜实验发现，在花粉管发育的整个时期，代表Ga蛋白的荧光均一的分布在整个质膜区域，尤其在尖端皮层区域荧光最亮，显示此处该蛋白浓度最高。无论是在正常发育的花粉管抑或是发生弯曲或扭曲生长的花粉管，均呈现同样的分布模式。随着花粉管发育，Ga蛋白表达量发生变化。在花粉管发育中期，Ga蛋白表达量比较高;随着花粉管离体培养时间的延长，Ga蛋白表达量下降。另外，在花粉刚刚萌发时，Ga蛋白表达量也比较低。 对白皮松花粉萌发进行的药理学实验显示，G蛋白调节剂 CTX和PTX对白皮松花粉管的影响呈现双阶段效应。当添加的药剂浓度小于400 ng mL-I时，无论CTX还是PTX均抑制了花粉萌发和花粉管生长，且花粉管容易破裂;而当二者浓度分别升至500 ng mL-I时，同对照相比，花粉管生长明显受到促进。这一结果不支持Ma等人在百合花粉中的研究结果。进一步应用FTIR技术分析发现，当用浓度为400 ng mL-I CTX或PTX处理花粉管时，花粉管细胞壁酚类物质增加，而纤维素、半纤维素、木聚糖等物质下降，这可能是导致此浓度处理下花粉管易破裂的原因。这些结果显示了G蛋白a亚基参与了白皮松花粉管生长，CTX和PTX可能通过下游对其敏感的功能蛋白而非Ga本身，影响着花粉管生长并调控着花粉管壁的建成。 利用来源于烟草的抗NtRacl抗体和拟南芥的抗ROPs抗体，应用WeternBlotting技术，我们在青杆花粉管中检测到分子量为23kDa的多肽。间接免疫荧光显微镜实验显示，在花粉萌发18和24小时后，Rac蛋白主要定位于花粉管尖端质膜区域，时而会延伸到顶端两侧区域，但从尖端到基部存在浓度梯度，这种分布模式多在花粉管发育的后期观察到。Rac蛋白在青杆花粉管不同发育时期的分布模式变化可能和花粉管的生长状态有关，在花粉管发育早期和中期，正是花粉管旺盛生长期，Rac蛋白的尖端定位保证了花粉管的极性生长。对Rac蛋白在花粉管的分布进行的连续切片扫描发现，Rac蛋白不但分布在质膜上，并与质膜偶联，而且在胞质中亦有分布。通过对一系列正常发育（即极性生长的花粉管）和畸形发育的花粉管进行观察发现，Rac蛋白主要分布在旺盛生长的花粉管尖端质膜或离顶端20 Vm处，在分叉的生长缓慢的分枝端分布较少。而在那些发生分叉生长的花粉管中，处于次要位置的基本停止生长的分枝端几乎没有Rac蛋白存在。在顶端发生膨大的花粉管中，Rac蛋白均匀分布在花粉管整个质膜上，丧失浓度梯度，失去极性生长。这些结果显示了Rac蛋白参与了青杆花粉管生长。 应用抗NtRacl抗体进行的间接免疫荧光显微镜定位实验，我们在正在生长的花粉管的管核中观察到明亮的荧光，显示了有Rac蛋白的存在。当精细胞在花粉粒中未移动到花粉管中时，几乎没有观察到荧光信号。随着花粉管发育，两个精细胞的位置发生变化，当其中一个较大的精细胞移动到花粉管中时，观察到明亮的荧光信号，这些结果显示了Rac蛋白可能参与了管核或精细胞在花粉管内的移动。

Identification of a novel gene K23 over-expressed in fish cross-subfamily cloned embryos

A novel gene-K23, differentially expressed in cross-subfamily cloned embryos, was isolated by RACE-PCR technique. It had 2580 base pairs (bp) in length, with a 1,425 bp open reading frame (ORF) encoding a putative protein of 474 amino acids (aa). Bioinformatic analysis indicated that K23 had 22 phosphorylation sites, but it had no signal peptides. Developmental expression analysis in zebrafish showed that K23 transcripts were maternally expressed in ovum and the amount of K23 transcripts increased gradually from zygote to pharyngula period. Subcellular localization analysis revealed that K23 protein was homogeneously distributed both in nuclei and cytoplasm. Taken together, our findings indicate that K23 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.